AAV-1â€“mediated gene transfer to skeletal muscle in humans

GENE THERAPY

AAV-1–mediated gene transfer to skeletal muscle in humans results indose-dependent activation of capsid-specific T cellsFederico Mingozzi,1 Janneke J. Meulenberg,2 Daniel J. Hui,1 Etiena Basner-Tschakarjan,1 Nicole C. Hasbrouck,1

Shyrie A. Edmonson,1,3 Natalie A. Hutnick,4 Michael R. Betts,4 John J. Kastelein,5 Erik S. Stroes,5 and Katherine A. High1,3

1Division of Hematology and Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, PA; 2Amsterdam Molecular Therapeutics,Amsterdam, The Netherlands; 3Howard Hughes Medical Institute, Philadelphia, PA; 4Department of Microbiology, University of Pennsylvania, Philadelphia; and5Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands

In a clinical trial for adeno-associatedvirus serotype 1 (AAV-1)–mediated genetransfer to muscle for lipoprotein lipase(LPL) deficiency, 1 subject from the high-dose cohort experienced a transient in-crease in the muscle enzyme creatinephosphokinase (CPK) 4 weeks after genetransfer. Simultaneously, after an initialdownward trend consistent with expres-sion of LPL, plasma triglyceride levelsreturned to baseline. We characterizedB- and T-cell responses to the vector and

the transgene product in the subjectsenrolled in this study. IFN-� enzyme-linked immunosorbent spot (ELISpot) andintracellular cytokine staining assays per-formed on peripheral blood mononuclearcells (PBMCs) from the subject who expe-rienced the CPK elevation showed theactivation of capsid-specific CD4� andCD8� T cells. Four of 8 subjects haddetectable T-cell responses to capsid withdose-dependent kinetics of appearance.Subjects with detectable T-cell responses

to capsid also had higher anti–AAV-1 IgG3antibody titer. No subject developed B- orT-cell responses to the LPL transgeneproduct. These findings suggest that T-cell responses directed to the AAV-1 cap-sid are dose-dependent. Whether theyalso limit the duration of expression ofthe transgene at higher doses is un-clear, and will require additional analy-ses at later time points. (Blood. 2009;114:2077-2086)

Introduction

Adeno-associated virus (AAV) vector–mediated gene transfer hasbeen successfully demonstrated in small and large animal mod-els,1-3 and translation of animal results into clinical studies iscurrently the major goal of the field. In a phase 1 study ofAAV-2–mediated gene transfer to liver in hemophilia B subjects,therapeutic levels of factor IX (FIX, � 10% normal) were achieved,but eventually fell to baseline (� 1%), accompanied by a transientand asymptomatic rise in liver enzymes that occurred simulta-neously with expansion of a population of circulating AAVcapsid–specific CD8� T cells.4,5 We hypothesized that this set offindings, observed in human subjects but not in animal models,2,6

arose from reactivation by vector infusion of a population ofcapsid-specific memory CD8� T cells generated originally inresponse to an infection by wild type AAV-2. The implicationsof these studies for AAV-mediated gene transfer have beenunclear, because comprehensive prospective studies of the im-mune response to capsid in AAV vector–injected human subjectsare lacking.

In the current study, we characterized the immune response toboth vector capsid and transgene product in a group of adultsubjects undergoing AAV-1–mediated gene transfer to skeletalmuscle for lipoprotein lipase (LPL) deficiency. Building on proof-of-concept studies in animal models,7,8 Stroes et al conducted anopen-label dose escalation study in which an AAV-1 vectorexpressing a naturally occurring variant of the LPL transgene(LPLS447X, a truncated version of the LPL protein associated withimproved lipid profile, carried by 20% of the general population7)

was introduced by direct intramuscular injection into the lowerextremities in subjects with LPL deficiency.9

Subjects were enrolled into 2 dose cohorts (n � 4 each),receiving either 1011 genome copies (gc)/kg or 3 � 1011 gc/kg;vector was administered by direct intramuscular injection aspreviously described.10 Vector administration was shown to be safeand well tolerated at all doses. Median plasma triglyceride (TG)initially decreased in all subjects, with 40% reduction in medianTG levels in 3 subjects, and detection of LPL transgene in biopsiesfrom injected muscle of 2 subjects from the high-dose cohort.However, long-term follow up of triglycerides showed loss ofefficacy in both dose cohorts after 18 to 31 months.9

In this study, we show that (1) none of the subjects demonstratedT-cell or B-cell responses to the LPL transgene product; (2) 4 of8 injected subjects showed a T-cell response to AAV-1 capsid aftervector injection, with kinetics that are dose-dependent; (3) 1 of8 subjects showed a rise in the muscle enzyme CPK (beginning 4 weeksafter vector injection) coinciding with an apparent loss of transgeneexpression, suggestive of T cell–mediated destruction of transducedmuscle cells; (4) 4 of 8 subjects, those with documented T-cell responsesto capsid, showed a rapid rise in anti–capsid IgG3 after vector injection,whereas the other 4 showed a slower, more modest rise. These resultsare consistent with previous findings of T-cell responses to capsid inhuman subjects undergoing hepatic gene transfer with anAAV vector,4,5

and extend the observations to a serotype other than AAV-2, with lowaffinity for heparin11 and another route of administration. Whether theseT-cell responses to capsid limit long-term transgene expression in some

Submitted July 8, 2008; accepted June 3, 2009. Prepublished online as BloodFirst Edition paper, June 8, 2009; DOI 10.1182/blood-2008-07-167510.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

2077BLOOD, 3 SEPTEMBER 2009 � VOLUME 114, NUMBER 10

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or all cases requires further investigation. If so, a general solution toovercoming host immune responses to gene therapy vectors may beneeded to reach the goal of long-term transgene expression in muscle orliver in human subjects.

Methods

Subjects

LPL-deficient subjects with missense mutations in both LPL alleles wereenrolled in the clinical trial; 8 subjects were enrolled in 2 dose cohorts(4 subjects per cohort) receiving 1011 gc/kg and 3 � 1011 gc/kg. Vector wasadministered intramuscularly into multiple sites at a dose of 1.6 to4.2 � 1011 gc/site of injection. The study was performed at the AcademicMedical Center, Amsterdam.9 High-resolution HLA typing of all subjectswas performed at the Department of Pathology and Laboratory Medicine,University of Pennsylvania Medical Center using the polymerase chainreaction sequence–specific primer kit Olerup by GenoVision and Life-MATCH kit (Tepnel Lifecodes) on a Luminex 100 detection system(Luminex Corp). Peripheral blood mononuclear cell (PBMC) isolation wasperformed by standard gradient techniques12 at the Academic MedicalCenter, Amsterdam; frozen PBMCs were sent to the Children’s Hospital ofPhiladelphia on dry ice. Experiments involving specimen collection ortesting were approved, respectively, by the Institutional Review Board andthe local Ethical Committees at the Children’s Hospital of Philadelphia andthe Academic Medical Center, and informed consent was obtained inaccordance with the Declaration of Helsinki.

Muscle biopsies analysis

Muscle biopsies were collected from subjects A, B, C, E, F, G, and Hbetween 10 and 36 weeks after gene transfer. Genomic DNA was isolatedfrom injected muscle tissue using the Gentra Puregene genomic DNApurification tissue kit (QIAGEN). One microgram of DNA was analyzed forAAV1-LPLS447X vector DNA sequence by quantitative polymerase chainreaction using primers and probe located in the boundary between the LPLcDNA and the WPRE element.

IFN-� ELISpot

A library of 146 15-mers overlapping in sequence by 10 amino acids wassynthesized (Mimotope) based on theAAV-1 VP1 amino acid sequence. Peptideswere arranged in a matrix of 24 pools, 12 to 14 peptides per pool. Recombinanthuman LPL protein resuspended in glycerol was used at 1.7 �g/mL, finalconcentration. Cytomegalovirus, Epstein-Barr virus, and influenza A–derived(CEF13) peptide pool (Mabtech) and a mix of phorbol 12-myristate 13-acetate(PMA) and ionomycin (Sigma-Aldrich) served as positive controls. In an IFN-�enzyme-linked immunosorbent spot (ELISpot) assay, PMA/ionomycin controlusually gives more than 1000 spot-forming units (SFU)/million cells, unless cellviability is below acceptable levels.

A positive response to an antigen or control was defined by several spotsper million PBMCs of at least 50 and at least 3 times the number of spotsmeasured for the medium only control.

T-cell responses were measured using one-color ELISpot assay forIFN-� (Mabtech) as previously described4,5; each antigen and control weretested in triplicate.

Depletion of CD4� or CD8� T cells was performed by magneticbead-conjugated antibodies according to the manufacturer’s protocol(Dynabeads; Invitrogen-Dynal). The fraction of PBMCs containing theuntouched CD4� or CD8� cells was used in an IFN-� ELISpot assay.

Perforin ELISpot

Perforin ELISpot was performed as previously described.14 Briefly, anti–humanperforin-precoated plates (Mabtech) were blocked for 1 hour at room temperaturewith AIM-V (Invitrogen-Gibco) containing 10% heat-inactivated FBS. Cellswere thawed, washed twice, and counted. PBMCs were plated at 2.5 � 105

cells/well in triplicate; test conditions included medium only, a pool of irrelevant

peptides (human retinal pigment epithelial 65 protein; Mimotopes) at a finalconcentration of 5 �g/mL per peptide, AAV-1 capsid particles at a finalconcentration of 40 �g/mL, and CEF (Mabtech) at a final dilution of 1:100,according to the manufacturer’s instructions.

PBMCs were incubated for 24 hours at 37°C, 5% CO2; afterwardincubation plates were washed 5 times with PBS and a biotin-conjugatedanti–human perforin antibody (Mabtech) was added to wells. Spot detectionwas performed using streptavidin-conjugated alkaline phosphatase (Mabtech)and BCIP/NBT substrate (KPL).

Intracellular staining for IFN-� and polyfunctional analysis ofT-cell responses

Intracellular cytokine staining for IFN-� was performed as previouslydescribed15; PBMCs were incubated at 37°C with 10 �g/mL peptide, or50 ng/mL PMA, 1 �g/mL ionomycin (Sigma-Aldrich), or medium alonefor 5 hours before surface and intracellular staining.

Polyfunctional analysis of T cells was performed as previously de-scribed.16,17 PBMCs were rested overnight at 2 � 106 cells/mL; thefollowing morning, cells were resuspended at 106 cells/mL with 1 �g/mLCD28 and CD29d antibodies and 10 �g/mL CD107a FITC. Fifteen-meroverlapping peptides covering the AAV capsid sequence were added to1 mL cells at a concentration of 2 �g/mL each. Cells were then incubatedfor 1 hour at 37°C and 5% CO2 before adding 1 �L per tube each ofbrefeldin A and Monensin (BD Biosciences). Cells were incubated for anadditional 5 hours before washing once in PBS. An Invitrogen aminereactive live/dead aqua dye was added to cells followed by surface stainwith CD3 Qdot 585, CD8 Texas Red PE, CD4 PeCy5.5, CD27 PeCy5,CD57 Qdot 565, CD45RO Qdot 705, CD14 PacBlue, CD16 PacBlue, andCD19 PacBlue. Cells were then stained intracellularly with IL-2 APC,IFN-� Alexa 700, perforin PE, and TNF- PeCy7 for 1 hour at roomtemperature. All antibodies were purchased from BD Biosciences; Quan-tum Dots were obtained from Invitrogen. Cells were run on a modified LSRII A (BD Biosciences) and analyzed using FlowJo 8.8 (TreeStar).

Anti-AAV capsid antibody assays

Anti–AAV-1 capsid Ig subclasses were measured with a capture assay;enzyme-linked immunosorbent assay plates were coated overnight at 4°Cwith 5 � 1010 capsid particles/mL AAV-1. Plates were blocked with 2%BSA, 0.05% Tween 20 in PBS for 2 hours at room temperature; serialdilutions of samples in blocking buffer were loaded and incubatedovernight at 4°C. Biotin-conjugated anti–human IgG1 (BD Biosciences),IgG2 (Sigma-Aldrich), IgG3 (Sigma-Aldrich), IgG4 (Sigma-Aldrich), orIgM (Sigma-Aldrich) was used as detecting antibodies at a dilution of1:4000. Immunoglobulin concentration was determined against standardcurves made with serial dilution of human purified IgG1 (Sigma-Aldrich),IgG2 (Sigma-Aldrich), IgG3 (Sigma-Aldrich), IgG4 (Millipore-Chemicon), or IgM (Sigma-Aldrich). Anti–AAV-1 neutralizing antibodytiter was determined as previously described.18

Bioinformatics and statistical analysis

A web-based bioinformatics tool19,20 was used to identify T-cell epitopes forthe HLA alleles of AAV-injected subjects. Statistical analysis of results wasperformed using GraphPad InStat version 3.0a (GraphPad Software).P values less than .05 were considered significant.

Results

Transient elevation of CPK following vector administration

Intramuscular administration of the AAV-1 vector encoding LPL(AAV-1-LPLS447X) proceeded uneventfully in all subjects.9 However, inthe first subject, subject E, receiving a dose of 3 � 1011 gc/kg, anincrease in CPK was observed beginning around week 4 and lasting forseveral weeks, returning to baseline by week 24 (Figure 1). Peak levelsof CPK were twice that of baseline and above the upper limit of normal

2078 MINGOZZI et al BLOOD, 3 SEPTEMBER 2009 � VOLUME 114, NUMBER 10




(190 IU/L). In this subject, plasma TG decreased in the first 4 weeksfollowing AAV-1- LPLS447X gene transfer, the expected result withsuccessful LPL expression. However the trend reversed beginning atweek 6, with kinetics similar to that of CPK levels (Figure 1), suggestinga relationship between the 2 phenomena. No CPK elevation wasobserved in any of the other subjects enrolled in the study with theexception of a transient elevation, still within the normal range, aroundweek 4 observed in subject H (supplemental Figure 1, available on theBlood website; see the Supplemental Materials link at the top of theonline article).

Activation of capsid T-cell responses demonstrates kineticsthat overlap with the rise and fall of serum CPK levels

PBMCs from subject E were isolated at baseline and after genetransfer. An IFN-� ELISpot specific for the AAV-1 capsid and theLPL transgene product did not show any reactivity at baseline,however beginning at week 4 and up to 12 weeks after gene transferT cells reacting to AAV-1 peptide pools became detectable, peakingaround week 6 at approximately 300 SFU/106 PBMCs (Figure 2).No B- or T-cell responses to the LPL transgene product weredetectable (data not shown, and9). T-cell responses returned tonegative by week 24 (Figure 2). Of note is the similarity in kineticsbetween the detectable T-cell response to AAV-1 capsid and theelevation in serum CPK (Figure 1).

Subject E’s HLA haplotype was obtained (supplemental Table 1)and used in a bioinformatics program to identify HLA class I and IIepitopes within the AAV-1 capsid VP-1 protein sequence predicted tobind to the subject’s alleles (Table 1); this analysis led to identification ofa 9-mer contained in peptide 145 (EPRPIGTRY), identified by IFN-�ELISpot (Figure 2B), binding to the HLA-B*1501 allele carried by thissubject; the 9-mer peptide was used in an IFN-� intracellular cytokinestaining assay with PBMCs isolated 6 weeks after gene transfer,confirming the presence of CD8� T cells reactive to this AAV-derivedepitope (Figure 3A-B).

In addition, a perforin ELISpot14 on subject E’s PBMCs wasused to confirm the cytolytic potential of CD8� T cells uponencounter of AAV capsid antigen but not when exposed to anirrelevant antigen or medium-only controls (Figure 3C). Themagnitude of response to AAV measured by perforin ELISpot wascomparable with the response directed against the CEF positivecontrol, a pool of MHC class I epitopes derived from cytomegalo-virus, Epstein-Barr virus, and flu antigens.

Administration of AAV-1-LPLS447X results in capsid-specificT-cell activation in 4 of 8 subjects with dose-dependent kinetics

Two of 4 subjects from the low-dose cohort (1011 gc/kg) and 2 of4 from the high-dose cohort (3 � 1011 gc/kg) had detectable T-cellresponses to capsid on an IFN-� ELISpot screening (Figure 4 andsupplemental Figure 2). Although the magnitude of response atweek 12 was similar among subjects (with the exception of subjectH, who had a weaker response; supplemental Figure 3), the kineticsof appearance of these responses differed between the 2 dosecohorts (Figure 4); subjects A and D, who received a low vectordose, had detectable T-cell responses to AAV capsid beginning atweek 12, whereas in subjects E and H the delay between vectoradministration and detection of a response by ELISpot was only4 and 6 weeks, respectively (Figure 4). Duration of detection ofT-cell responses also differed between the 2 cohorts of subjects,low-dose cohort subjects having detectable responses up to week36, and high-dose cohort subjects only up to week 12 (Figure 4).Long-term follow up of capsid T-cell responses by IFN-� ELISpot(up to 24 months for some of the subjects) confirmed persistence ofthese responses only in subjects A and D from the low-dose cohort(data not shown).

In contrast to results of anti–AAV-1 neutralizing antibody assay(supplemental Table 3), no subject had detectable T-cell responsesto the AAV-1 capsid before vector administration, confirming thatprimed capsid-specific T cells, if present prior to vector injection,are not easily detectable in PBMCs.4,5

None of the subjects enrolled in the clinical study had detectableB- or T-cell responses to the LPL transgene9 (and data not shown)before or after vector administration. Vector genome copy numberwas evaluated on muscle biopsies collected between weeks 10 and36. Notably, vector genome copy number was lower in subject Ecompared with subjects F and G from the same dose cohort (Table2); analysis of the biopsy in subject H showed no detectable levelsof vector genomes, indicating either complete clearance of trans-duced muscle fibers or, alternatively, that the injected area wasmissed during muscle biopsy.

Identification of MHC class I and II epitopes within the AAV-1capsid sequence

A matrix of 24 pools of 15-mers overlapping by 10 amino acidsspanning the entire sequence of AAV-1 VP-1 capsid protein was

TGCPK

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TRIGLYCERIDE RANGE PRIOR TO GENE TRANSFER

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Figure 1. Plasma TG and serum CPK levels in subjectE. Vertical dotted line represents time of vector adminis-tration; TG levels are expressed in millimoles per liter(mM) and CPK in international units per liter (IU/L).*Positive capsid IFN-� ELISpot. CPK: ULN 190 IU/L; TG:normal levels less than 2 mM, LPL study inclusion criteriamore than 10 mM. Mean ( SD) TG prior to gene transfer(week �42 to 0) for subject E was 42.7 mM ( 9.3 mM)(shaded area).

T-CELL RESPONSES TO AAV-1 IN HUMAN SUBJECTS 2079BLOOD, 3 SEPTEMBER 2009 � VOLUME 114, NUMBER 10




used to screen PBMCs collected from all subjects enrolled in theclinical study (Figure 2B). For each subject, ELISpot results(supplemental Figure 2) were matched with the high-resolutionHLA haplotype (supplemental Table 2) to identify class I and IIpeptide epitope candidates (Table 1). CD4� and CD8� depletion,followed by IFN-� ELISpot assay, was used to confirm thesubset of T cells responding to the identified epitopes (Table 1);interestingly, subjects A and D, who were enrolled in the lowvector dose cohort and had a delayed detection of capsid T-cellresponses in PBMCs, showed predominantly a CD4� T-cellresponse (Figure 5) in contrast to subjects E and H, in whom

both CD4� and CD8� T-cell responses were observed (Figure 3and Table 1).

AAV capsid administration results in the activation of bothCD4� and CD8� T cells with production of TNF-�

Phenotypic and functional analysis of T-cell responses was per-formed by multicolor flow cytometry. PBMCs collected at baselineand after gene transfer were restimulated in vitro with peptidesderived from the AAV capsid and stained for T-cell activationmarkers. In all subjects, upon vector administration, activation of

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Pool 13 Pool 14 Pool 15 Pool 16 Pool 17 Pool 18 Pool 19 Pool 20 Pool 21 Pool 22 Pool 23 Pool 24

Pool 1 1 2 3 4 5 6 7 8 9 10 11 12

Pool 2 13 14 15 16 17 18 19 20 21 22 23 24

Pool 3 25 26 27 28 29 30 31 32 33 34 35 36

Pool 4 37 38 39 40 41 42 43 44 45 46 47 48

Pool 5 49 50 51 52 53 54 55 56 57 58 59 60

Pool 6 61 62 63 64 65 66 67 68 69 70 71 72

Pool 7 73 74 75 76 77 78 79 80 81 82 83 84

Pool 8 85 86 87 88 89 90 91 92 93 94 95 96

Pool 9 97 98 99 100 101 102 103 104 105 106 107 108

Pool 10 109 110 111 112 113 114 115 116 117 118 119 120

Pool 11 121 122 123 124 125 126 127 128 129 130 131 132

Pool 12 133 134 135 136 137 138 139 140 141 142 143 144

145 146

A

B

Figure 2. Capsid-specific IFN-� ELISpot on PBMCs from subject E. (A) Capsid-specific IFN-� ELISpot; results are expressed in SFU/106 PBMCs (average SD). Errorbars represent SD. P 1 to P 24, AAV peptide matrix pools. Positive pools are indicated (positive defined as at least 3-fold above the medium control and at least 50 SFU/106

PBMCs). Horizontal lines represent the cutoff for positivity. PMA indicates positive control (� 1000 SFU/106 PBMCs for all time points); M, medium only. (B) Matrix analysis ofresults. Peptides identified by positive pools at any time point are indicated in black boxes. Note that peptides 145 and 146 were included in the matrix pools 12 and 13.





CD4� and, to a lesser extent, CD8� T cells was observed withproduction of TNF- (Figure 6A) in addition to IFN-�. Phenotypicanalysis showed a mix of naive (CD27�CD45RO�) and memory(CD27�CD45RO�) CD4� T cells reacting to the AAV capsid(Figure 6B). The polyfunctional analysis of T-cell responses to theAAV capsid is summarized in Figure 7; pie charts show the degreeof T-cell activation as measured by the number of T-cell activationand functional markers (IL-2, IFN-�, perforin, TNF-, and CD107)expressed by CD4� or CD8� T cells. Notably, some level of

reactivity of CD4� and CD8� T cells was measured at baselinebefore vector administration. This could be accounted for bypre-exposure to wild-type AAV, as documented by baseline neutral-izing antibody titer (supplemental Table 2). The highest level ofactivation (expression of 4/5 activation markers upon restimulationwith AAV-1 peptides) was measured for subject E’s CD8� T cellsin PBMCs collected at 6 and 12 weeks after gene transfer; details ofthe polyfunctional analysis of T-cell responses to capsid in subjectE are shown in supplemental Figure 3.

Table 1. AAV-1 peptide epitopes identified

Peptides identified by IFN-� ELISpot Amino acid position Peptide library pools Peptide library no. HLA binding prediction MHC restriction

Subject A

SSFYCLEYFPSQMLRTGNNF 390 7-19/20 79/80 N/D II†

Subject D

TTSTRTWALPTYNNH 247 5-13 49 HLA-DR*0801 II†

TTSTRTWALPTYNNH 243 5-13 49 HLA-DR*1302 II†

Subject E

TTSTRTWALPTYNNH 240 5-13 49 N/D II†

YTEPRPIGTRYLTRP 724 12-13 145 HLA-B*1501 I‡

EFSATKFASFITQYS 665 12-13 133 HLA-DR*1501 II

Subject H

VQVFSDSEYQLPYV 341 6-21 69 HLA-A*3301 I

Bold, underlined amino acid sequences represent the epitopes predicted using online MHC-binding prediction programs.19,20 Amino acid position indicates the position ofthe first amino acid of the epitope in the AAV-1 VP1 capsid protein sequence. MHC restriction indicates whether CD8� (class I) or CD4� (class II) T cells recognize the epitopesidentified.

N/D indicates not determined.†Confirmed by IFN-� ELISpot assay.‡Confirmed by IFN-� intracellular staining.

Figure 3. Functional characterization of capsid-specific T cells. (A) Intracellular IFN-� staining on subject E’s week-6 PBMCs; numbers indicate the percentage ofCD4�CD8� T cells that are IFN-��. Cells are gated on forward and side scatter, on singlets, and on CD4�CD8� T cells. (B) Representation of 3 independent intracellular IFN-�staining experiments (average SD of % CD4�CD8�IFN-�� T cells). (C) Perforin ELISpot on subject E’s PBMCs. Medium indicates medium only control; irrelevant, humanretinal pigment epithelium 65 protein; AAV-1, AAV-1 capsids; and positive, CEF peptide pool. Data are expressed in SFU per 106 PBMCs. SFU per 106 PBMCs were comparedbetween AAV-1 and medium control (P � .008), AAV-1, and irrelevant control (P � .007), and AAV-1 and positive controls (P � .05, not significant).





Subjects with a detectable IFN-� response to AAV capsiddevelop higher titer of anti–AAV-1 IgG3 antibodies

No clear correlation between the titer of pre-existing neutralizingantibodies to the AAV-1 capsid and the detection of T-cellresponses to the capsid after gene transfer was detected (supplemen-tal Figure 2) in agreement with previous findings in the context of astudy for AAV-2–mediated hepatic gene transfer in severe hemo-philia subjects.4

Anti–AAV-1 IgG1, IgG2, IgG3, and IgG4 subclasses and IgMwere determined. Antibody titer for IgG subclasses and IgM rose inall subjects upon vector infusion; subjects with positive capsidIFN-� ELISpot had statistically significantly higher (P � .05 atday 28 after injection) anti–AAV-1 IgG3 titers (Figure 8).

Discussion

The potential of AAV vectors as a therapeutic tool has beenestablished in several preclinical models of disease.1,21-23 In retinalgene transfer, compelling data in dog models of inherited blindnessscaled up uneventfully to human subjects.24,25 However, recentfindings in a clinical study for hepatic gene transfer with an AAV-2vector expressing coagulation FIX in severe hemophilia B subjectshighlighted an issue previously unidentified in experimental ani-mals. In this study, 2 subjects developed a transient, self-limitedelevation of liver enzymes following gene transfer; concomitantly,

a population of capsid-specific CD8� T cells expanded and con-tracted with similar kinetics, suggesting immune destruction oftransduced hepatocytes.4,5 Our hypothesis is that pre-existingmemory T cells to the AAV capsid are responsible for the loss oftransgene expression observed in humans but not in experimentalanimals.5

The present study represents the first complete prospectivelyanalyzed characterization of B- and T-cell responses to the AAV

Positive IFN-γ ELISpot to AAV-1Negative IFN-γ ELISpot to AAV-1

0 2 4 6 8 12 16-26 39-48

A

B

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D

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G

H

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***

Figure 4. Time course of T-cell responses to AAV-1 capsid measured by IFN-�ELISpot. Each horizontal bar represents an individual subject; time after intramuscu-lar administration (in weeks) is indicated by the vertical lines. Gray bars indicate anegative ELISpot result; red bars indicate a positive ELISpot result. A time point wasconsidered positive when the average SFU/106 PBMCs was higher than 3 � themedium control and at least 50 SFU/106 PBMCs for both the AAV-1 peptide pools andthe AAV-1 empty particles antigens. The vector dose, in genome copies per kilogram(gc/kg), received by the subjects in the 2 cohorts is indicated on the left of the graph.*Negative for AAV-1 empty capsids.

Table 2. Vector genome copy number per diploid genome in musclehomogenates from AAV1-LPL–injected subjects

SubjectWeeks after vector

administrationVector genome copy no.,

gc/diploid genomes

A 36 0.37

B 36 0.03

C 28 1.08

D na na

E 10 1.14

F 32 5.58

G 26 14.4

H 28 und

na indicates not available, subject withdrew consent; and und, undetectable.

Figure 5. CD4� and CD8� T-cell depletion experiments. IFN-� ELISpot onPBMCs, or CD4- or CD8-depleted fraction of PBMCs (CD4�CD8� or CD4�CD8�,respectively). Samples were collected at week 19 (subject A) and week 12 (subjectD). Results are expressed in SFU/106 cells as average ( SD) of 3 replicates.Peptide 79/80 indicates 15-mers previously identified in subject A by screeningELISpot; peptide 49, 15-mer previously identified in subject D by screening ELISpot;medium, negative control; and PMA, positive control (PMA and ionomycin).

Figure 6. T-cell response to AAV capsid proteins. PBMCs were stimulated for6 hours with a set of 15-mer peptides covering the AAV capsid protein sequence andcytokine responses assessed by polychromatic flow cytometry. (A) TNF- responseto capsid proteins in CD4� and CD8� T cells. Numbers in the top right corner indicatethe percentage of responding CD4� or CD8� T cells. A minimum of 240 000 eventswere collected for each time point. (B) Immunophenotyping of responding TNF-�

cells (red dots) compared with total CD4� T cells (gray density plots). Cells weregated on forward and side scatter, CD3� cells, CD14�CD16�CD20� cells. Cells werealso stained with an Invitrogen amine reactive live/dead aqua dye.





capsid and to the transgene product in a clinical trial of AAV-mediated gene transfer.

Several conclusions can be drawn from this study. First, subjectE is of special interest. The constellation of findings in his case, aninitial approximately 40% drop of serum triglyceride levels,followed by a return to baseline coincident with a rise in the muscleenzyme CPK between weeks 4 and 12, is suggestive of a process inwhich transduced muscle fibers (expressing LPL) are destroyed.The fact that these events overlap chronologically with theactivation of T-cell responses to AAV capsid (as measured byIFN-� ELISpot), and that gene copy number in subject E isconsiderably lower than F and G, the 2 subjects who received thehigher dose of vector and did not have detectable capsid T-cellresponses, is also consistent with destruction of transduced cells,and moreover suggests that these phenomena may be linked. Thefact that residual levels of muscle transduction were still detectable

in subject E may be accounted for by the timing of muscle biopsy,which occurred at week 10 after vector injection for subject E.A biopsy at a later time point would be required to determinewhether the donated DNA had persisted.

The lack of evidence of T-cell responses to the transgeneproduct is another finding shared by both the AAV-2 intrahepaticgene transfer for hemophilia and the AAV-1 LPL intramusculargene transfer clinical studies. Based on animal studies,2,26 theabsence of a response to the transgene product would be predictedfor liver-directed gene transfer, which tends to promote tolerance tothe transgene product,27-29 but not necessarily for muscle-directedgene transfer, where immune responses to the transgene producthave more commonly occurred.30,31 The absence of immuneresponses is likely at least partly accounted for by the exclusionfrom this study of all subjects except those with a missensemutation as the cause of LPL deficiency, that is, the study

Figure 7. Polyfunctional analysis of T-cell responsesto the AAV-1 capsid. Concurrent expression of IL-2,IFN-�, perforin, TNF-, and CD107 is measured atbaseline and after gene transfer. Pie charts represent theproportion of capsid-specific CD4� (left) or CD8� (right)T cells expressing 1, 2, 3, 4, or 5 markers. Cells weregated on forward and side scatter, CD3� cells,CD14�CD16�CD20� cells. Cells were also stained withan Invitrogen amine reactive live/dead aqua dye. CD4� orCD8� T cells were analyzed for IL-2, IFN-�, perforin,TNF-, and CD107 expression in response to AAV-1peptides against medium control.





was limited to subjects with some degree of tolerance to thetransgene product.

It is important to note that, at the levels of muscle enzymeelevation reported here, and the levels of liver enzyme elevationreported in the AAV-2-FIX trial, these are safety signals, rather thanadverse events, because the changes were short-term and resolvedwithout medical intervention. Tissues such as liver and skeletalmuscle are capable of regeneration after damage, an advantage notshared by certain other target tissues of interest including cardiacmuscle, where there is limited capacity to regenerate after damage.This highlights the critical nature of a careful analysis of thisproblem before dose escalation in more vulnerable tissues.

The analysis of the subjects in this trial highlights other findingsof interest. First is that administration of higher doses of vector toskeletal muscle results not in a higher readout on the IFN-�ELISpot, but in a faster generation of a detectable immuneresponse to capsid. This may have important clinical consequences,based on the current understanding of the fate of the capsid aftervector transduction of the target cells. If, as has been proposed,transduced cells are characterized by presentation of capsid anti-gens via MHC class I, then the cells would be vulnerable todestruction by cognate CD8� T cells for as long as capsid ispresented on the cell surface; a recent study32 showing up-regulation of MHC class I expression on muscle fibers after AAV-1gene transfer supports this hypothesis. However, immune re-sponses that develop slowly and do not become detectable untillater time points33 may in fact encounter few or no targets forimmune-mediated destruction.

These data also suggest that there may be a dose dependence todevelopment of a CD8� T-cell response, based on the finding thatfurther analysis of the positive IFN-� ELISpots in the low-dosesubjects indicated that these are driven by CD4� rather than CD8�

T-cell epitopes. This is in contrast to the data from the higher dosegroup, where intracellular cytokine staining, perforin ELISpot, and

bioinformatics analysis of the epitopes identified are indicative of amixed CD4� and CD8� T-cell–mediated immune response to theAAV capsid.

Not surprisingly, the administration of an AAV-1 vector resultsin activation of both CD4� and CD8� T cells; CD4� T-cellactivation is required for helper functions both in humoral andcytotoxic responses. Furthermore, it is clear that CD4� T cells playan important role in disease control in viral infections,34 where thispopulation of virus-specific T cells shows predominantly produc-tion of IFN-� and TNF-35 as we documented here. Successfulimmune response to a virus is characterized by a complex patternof activation, and looking only at IFN-� may underestimate thetotal antigen-specific CD8� T-cell frequency.36

Consistent with previous population studies,5,37 PBMCs of all8 subjects were negative for responses to AAV capsid by IFN-�ELISpot before vector infusion, independent of pre-exposure towild-type AAV as indicated by anticapsid neutralizing antibodiesmeasured at baseline. Moreover, even in those who developed aresponse after vector infusion, the duration of detectable responsewas quite short in some (eg, subject E), suggesting that(1) screening of PBMCs will not be useful for predicting whichsubjects are likely to develop a T-cell response to capsid; and(2) capsid-specific CD8� T cells, if already present, likely reside incompartments other than the peripheral blood. For example, in anearlier study we were able to expand capsid-specific T cells fromup to 60% of subjects when lymphocytes isolated from the spleenwere used as starting material.5

Interestingly, the average IgG3 titer of subjects who haddetectable IFN-� responses to the AAV capsid was higher than therest of the subjects. IgG3 together with IgG1 are involved inhumoral responses to acute infections with parvovirus B19,38 andare the prevalent IgG subclasses in diseases characterized by IFN-�response.39 Whether IFN-� helps to drive an immunoglobulin classswitch under these circumstances is unknown. It should be noted

Figure 8. Anti–AAV-1 antibody subclass analysis. Serum levels of IgG1, IgG2, IgG3, IgG4, and IgM (average SD, ng/mL). � indicates subjects with positive T-cellresponse to the AAV capsid measured by ELISpot (subjects A, D-E, and H); u, subjects with no detectable T-cell response to the AAV capsid measured by ELISpot (subjectsB-C, F-G); *P � .05.





also that IgG1 and IgG3 are involved in antibody-dependentcell-mediated cytotoxicity, although it is not clear whether antibody-dependent cell-mediated cytotoxicity played a role in the findingsnoted here.

It is perhaps critical to underscore the dose dependence of theT-cell response with AAV vectors, with 2 points deserving empha-sis. Injection of low doses of vector, such as occurs in early phasegene transfer for genetic disease, or in vaccine studies, is not likelyto result in capsid-specific T-cell activation. Thus presence orabsence of T-cell activation should be carefully correlated with thedoses being injected. Second, the findings have implications forvector design; vectors that gain access to target cells moreefficiently may result in increased presentation of antigen to theimmune system, and more efficient activation of T cells.

Improvements in the expression cassette itself, which allowgreater protein expression per introduced virion, are likely todecrease the vector dose required and thus lessen the risk foractivation of circulating capsid-specific T cells. Thus more efficientserotypes may enhance risk, whereas more efficient expressioncassettes will reduce it.

At a dose of 1.6 to 4.2 � 1011 gc per site of injection, the AAV-1vector used in this study resulted in muscle transduction at a levelof 0.5 to 7.0 vector gene copies per diploid genome and evidencefor a capsid-specific T-cell response in 4 of 8 subjects; differently,in an AAV-2 muscle gene transfer study for hemophilia B, a dose of1.5 � 1012 gc per site of injection gave 0.5 to 4.0 vector genecopies per diploid genome, no apparent T-cell response to capsidand long-term expression of transgene.10,40 The 3- to 10-fold higherperformance of AAV-1 over AAV-2 in transducing muscle, leadingto a higher capsid antigen load and thus better antigen presentation,may explain the different immunogenic profiles of the 2 serotypes.

Continuing dose escalation may address the question as towhether responses are detected in greater numbers of subjects athigher doses. Alternatively, previous exposure to wild-type AAVmay account for the different outcomes of gene transfer in subjectsin this study; the percentage of positive T-cell responses observedhere is consistent with the observation that approximately 50% ofthe population is positive for anti-AAV antibodies.5 Polyfunctionalanalysis of T-cell responses to capsid in this study revealed somebaseline levels of CD4� and CD8� activation, which, together withthe memory phenotype exhibited by AAV-specific T cells, isconsistent with this last hypothesis. It is also important to note thatexamining circulating PBMCs is only a surrogate for examiningT cells in the target tissues, so that the readout here mightunderestimate numbers of subjects with T-cell infiltrates in injectedmuscle.

An unanswered question regarding these findings is whetherT-cell responses predict clinical outcomes. The relationship of

T-cell responses, changes in muscle enzymes, and long-termexpression of the donated gene requires further study. Recentstudies in mice and humans32,41 showed apoptosis of lymphocytesinfiltrating skeletal muscle after AAV-mediated gene transfer, aphenomenon that may play a role in shaping clinical responses toT-cell activation, and may also account for variability of responsesamong subjects receiving equivalent vector doses.

In summary, we demonstrated that the intramuscular administra-tion of an AAV-1 vector in humans results in T-cell activation in 4of 8 subjects. The results here extend previous observations madein the context of an AAV-2 administered intravascularly in hemo-philia B subjects to another serotype, AAV-1, with low affinity toheparin,11 another target tissue, and another route of administration.Whether these T-cell responses limit long-term expression in everycase is not yet clear. Potential solutions such as the use of transientimmunomodulation around the time of gene transfer may berequired to achieve sustained, persistent expression of the trans-gene product.

Acknowledgments

This work was supported by National Institutes of Health (NIH)grant P01 HL078810 to K.A.H. and the Howard Hughes MedicalInstitute. N.C.H. was supported by training grant NIH T32HL007150, and D.J.H. by training grant NIH T32 HL07439. TheAAV-lipoprotein lipase clinical study was supported by AmsterdamMolecular Therapeutics.

Authorship

Contribution: F.M. designed and performed the experiments, anddrafted the paper; J.J.M. coordinated sample collection, partici-pated in experimental design, and drafted the paper; D.J.H.,E.B.-T., N.C.H., and S.A.E. participated in experimental activities;N.A.H. performed multifunctional analysis of T cells; M.R.B.provided critical insights to paper preparation; J.J.K. and E.S.S.performed clinical work; and K.A.H. supervised experimentaldesign and execution, performed data analysis, and draftedthe paper.

Conflict-of-interest disclosure: F.M., D.J.H., and K.A.H. holdpatents related to AAV gene therapy. The remaining authors declareno competing financial interests.

The current address for Dr Meulenberg is ORCA Therapeutics,Amsterdam, The Netherlands.

Correspondence: Katherine A. High, The Children’s Hospital ofPhiladelphia, ARC Suite 302, 3615 Civic Center Blvd, Philadel-phia, PA 19104; e-mail: [email protected].

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online June 8, 2009 originally publisheddoi:10.1182/blood-2008-07-167510

2009 114: 2077-2086

Stroes and Katherine A. HighHasbrouck, Shyrie A. Edmonson, Natalie A. Hutnick, Michael R. Betts, John J. Kastelein, Erik S. Federico Mingozzi, Janneke J. Meulenberg, Daniel J. Hui, Etiena Basner-Tschakarjan, Nicole C. dose-dependent activation of capsid-specific T cells

mediated gene transfer to skeletal muscle in humans results in−AAV-1

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